1. Field of the Invention
The present invention relates to a confocal microscope, and more particularly, to a method for adjusting the confocal microscope, which eliminates occurrence of synchronous fringes (moiré fringes) during observation of a sample.
2. Description of the Related Art
A confocal microscope is for observing a sample by scanning a point of collected light on the sample through use of a laser (hereinafter called as a light beam for optical image measurement) and making fluorescent light that returns from the sample form an image. The confocal microscope is used for observing physiological reaction and morphology of live cells in the fields of biology, biotechnology, and the like, or for observing the surface of an LSI (large scale integration) in the field of semiconductors, etc.
FIG. 1 is a block diagram of a principal section showing an example of a confocal microscope used in the related art and in an embodiment of the present invention. In FIG. 1, a confocal scanner unit 1 is connected to a port 3 of a microscope unit 2, and a light beam 4 for image measurement is collected to individual light fluxes by microlenses 6 of a microlens array disk 5. After having passed through a dichroic mirror 7, the light fluxes pass through individual pinholes 9 of a pinhole disk (hereinafter called as a Nipkow disk) 8, and are collected onto a sample (omitted from the drawings) on a stage 11 by an objective lens 10 of the microscope unit 2.
The sample gives fluorescent light by radiation of the light beam 4 for image measurement. The fluorescent light returned from the sample again passes through the objective lens 10, and is collected onto the individual pinholes 9 of the Nipkow disk 8. The returned fluorescent light having passed through the individual pinholes is reflected by the dichroic mirror 7, and is imaged by an image sensor 13 (CCD: charge coupled device) via a relay lens 12.
In such an apparatus, the microlens array disk 5 and the Nipkow disk 8 are coaxially rotated at a given speed by a motor which is not shown, and the point of collected light on the sample is scanned by a movement of the pinholes 9 induced by the rotation.
The surface of the Nipkow disk 8 where the pinholes 9 are arranged, a surface to be observed on the sample, and a light-receiving face of the image sensor 13 are arranged in an optically conjugate relationship. Accordingly, an optical cross-sectional view of the sample, namely, a confocal image, is formed on the image sensor 13. Details of the confocal microscope of a Nipkow disk type are described in JP-A-5-60980.
JP-A-5-60980 describes a confocal optical scanner, in which imaging characteristic is enhanced and stray light from the pinhole surface is reduced.
Incidentally, in such a confocal microscope, the pinholes thoroughly scan over the screen that is displayed via the image sensor to create one screen every time the pinhole disk rotates for 30°.
For instance, when the disk is rotating at 5000 rpm, the time required to create one screen is computed as 1 ms in accordance with the following equation; specifically,5000 rpm/60 seconds=83.33 rpsFrame rate=83.33*12=1000 fps (1000 frames per second)Frame cycle= 1/1000 fps=1 ms
During observation of an image by using the confocal microscope, when an exposure time of the camera becomes shorter (100 ms or less, particularly, a video rate of 33 ms or less), circular-arc lateral fringes called synchronous fringes, such as those shown in FIG. 2A, sometimes appear in an image. The reason for this is that, in a case where one image is formed when the Nipkow disk has rotated for 30°, for example, a phase difference arises between the exposure time of the CCD digital camera and the rotation of the disk.
There are an equiangular spiral layout, a square layout, a constant pitch spiral layout and the like, as ways of arranging pinholes. The arrangement of the pinholes used in the present invention is the constant pitch spiral layout such as that shown in FIG. 2B.
When an NTSC (National Television System Committee) camera is used, there is prepared a mechanism for inputting a video composite signal of the camera to a synchronous input terminal of the confocal microscope to thus synchronize the rotation of a disk. However, in the case of a CCD digital camera which performs non-interlace imaging, an effective mechanism for synchronizing the rotation of the disk is not available.